Introduction

A. The Use and Choice of Selectable Markers

One of the major problems with current approaches to gene therapy is the instability of expression of genes transferred into recipient cells. Although in theory, homologous recombination or use of artificial chromosomes can stabilize sequences with wild-type regulatory regions, such approaches to gene therapy are not yet feasible and may not be efficient for some time to come. In most high efficiency DNA transfer in current use in intact organisms, selectable markers must be used to maintain transferred sequences; in the absence of selection the transferred DNAs or their expression is rapidly lost.

There are several different selectable markers that might be used for in vivo selection, including genes whose expression has been associated with resistance of cancers to anticancer drugs. Examples include: (a) methotrexate resistance due to mutant dihydrofolate reductase [DHFR] (1); (b) alkylating agent resistance due to expression of methylguanine methyl-transferase [MGMT] (2); and (c) the expression of the multi-drug transporting proteins P-glycoprotein (P-gp, the product of the MDR1 gene) (3) and MRP (multidrug resistance associated protein) (4). In this chapter, we will detail our experience with the MDR1 gene.

Current affiliation:

1 Technical University of Munich, Munich, Germany.

2 Cellular Genomics, Inc., Branford, Connecticut, U.S.A.

3 National University of Singapore, Singapore.

4 QBI Enterprises Ltd., New Ziona, Israel.

5 University Hospital Freiburg, Freiburg, Germany.

6 Purdue University, West Lafayette, Indiana, U.S.A.

The resistance of many cancers to anticancer drugs is due, in many cases, to the overexpression of several different ATP-dependent transporters (ABC transporters), including the human multidrug resistance gene MDR1 (ABC B1) (3,5,6), MRP1 (ABC C1), the multidrug resistance-associated protein (7) and other MRP family members (8), and MXR (ABC G2) (9). MDR1 encodes the multidrug transporter, or P-glycopro-tein (P-gp). P-gp is a 12 transmembrane domain glycoprotein composed of 2 homologous halves, each containing 6 transmembrane (TM) domains and one ATP binding/utilization site. P-gp recognizes a large number of structurally unrelated hydrophobic and amphipathic molecules, including many chemotherapeutic agents, and removes them from the cell via an ATP-dependent transport process (see Fig. 1).

MDR1 has many obvious advantages for use as a selectable marker in gene therapy. It is a cell surface protein that can be easily detected by FACS or immunohistochemistry. Cells expressing P-gp on their surfaces can be enriched using cell sorting or magnetic bead panning technologies. The very broad range of cytotoxic substrates recognized by P-gp makes it a pharmacologically flexible system, allowing the investigator to choose among many different selection regimens with differential toxicity for different tissues and different pharma-cokinetic properties. Furthermore, as will be discussed in detail in this chapter, P-gp can be mutationally modified to increase resistance to specific substrates and alter inhibitor sensitivity. Hematopoietic cells initially appeared to tolerate relatively high levels of P-gp expression without major effects on differentiated function (10).

B. Lessons from Transgenic and Knockout Mice

Two lines of evidence support the concept of using MDR1 as a selectable marker in human gene therapy. Transgenic mice

Figure 1 Multidrug transporters such as MDR1, MRP1, and MXR confer resistance on transduced cells. The triangles are cytotoxic drugs; the large, bold arrows are transporters.

expressing the MDR1 gene in their bone marrow are resistant to the cytotoxic effects of many different anticancer drugs (10-12). MDR1 transgenic bone marrow can be transplanted into drug sensitive mice, and the transplanted marrow is resistant to cytotoxic drugs (13). Mice transplanted with bone marrow transduced with the human MDR1 cDNA and exposed to taxol show specific enrichment of the MDR1-transduced cells (14-16), and this transduced marrow can be serially transplanted and remains drug resistant (16). Recently, this ability to select transduced bone marrow with taxol has been demonstrated in a canine bone marrow transplantation model (17).

The mouse mdr1a and mdr1b genes have been insertionally inactivated in mice (18-21). These animals, although otherwise normal, are hypersensitive to cytotoxic substrates of Pgp. This hypersensitivity is due in part to the abrogation of the mdr1a-based blood brain barrier (22), and to enhanced absorption and decreased excretion of mdr1 substrates (23). These studies demonstrate the critical role that P-gp plays in drug distribution and pharmacokinetics, and argue that specific targeting of P-gp to tissues that do not ordinarily express it (as in gene therapy), will protect such tissues from cytotoxic mdr1 substrates.

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment